Combined Textile Abrading And Color Modification

ABSTRACT

Described are compositions and methods for the enzymatic abrading and color modification of dyed textiles. The compositions and methods permit a textile manufacturer to obtain a wide variety of different textile finishes and colors using exclusively enzymatic methods.

PRIORITY

The present application claims priority to U.S. Provisional ApplicationSer. Nos. 61/237,534, filed on Aug. 27, 2009, and 61/238,029, filed onAug. 28, 2009, which are hereby incorporated by reference in theirentirety.

TECHNICAL FIELD

The present compositions and methods relate to combined enzymatictextile abrading and color adjustment. The composition and methods arebased, in part, on the discovery that certain enzymes can be usedsequentially, sometimes in the same treatment bath, to produce textileswith a broad range of finishes and colors using only a limited suite ofenzymatic systems.

BACKGROUND

The use of enzymes to process textiles is now well established. Amylasesare used for desizing, cellulases are used for abrading and abrading,and catalases are used for bleach clean-up. More recently, enzymes suchas perhydrolases and laccases have been applied to textile processing,where such enzymes are used in place of harsh chemical bleachingtreatments.

Although enzymatic textiles treatments have greatly reduced theenvironmental impact of textile processing and produced significant costsaving to textiles producers, the complete manufacture of a textileproducts continues to require multiple discrete steps, frequentlyinvolving separate baths and multiple rinse cycles to remove thereaction components from one process prior to initiating a subsequentprocess. In addition, enzymatic textile processing has heretofore notbeen capable of producing the array of finishes and colors demanded bymodern textile consumers, thereby limiting it acceptance.

SUMMARY

Compositions and methods relating to combined enzymatic textile abradingand color adjustment are described.

In one aspect, an enzymatic method for abrading and modifying the colorof a dyed textile is provided, comprising: (a) contacting the textilewith a cellulase to biopolish the textile; and (b) contacting thetextile with a perhydrolase enzyme system to modify the color of thetextile; wherein (a) and (b) are performed in a single bath. In someembodiments, (a) and (b) are performed sequentially or simultaneously.

In some embodiments, (a) is preceded by an enzymatic desizing step,which may be performed in the same bath as (a) and (b). In someembodiments, (b) is followed by the addition of a catalase enzyme, whichmay be added to the same bath in which (a) and (b) are performed.

In another aspect, an enzymatic method for abrading and modifying thecolor of a dyed textile is provided, comprising: (a) contacting thetextile with a composition comprising a cellulase to abrade the textile;(b) contacting the textile with a laccase enzyme system to perform afirst color modification of the textile; and (c) contacting the textilewith a perhydrolase enzyme system to perform a second color modificationof the textile; wherein the overall color modification produced by thecombination of (b) and (c) is different from the first colormodification in (b) and the second color modification in (c).

In some embodiments, (b) is performed before (c). In some embodiments,(a) and (b) are performed sequentially or simultaneously in a singlebath.

In some embodiments, (c) is performed before (b). In some embodiments,(a) and (c) are performed sequentially or simultaneously in a singlebath. In some embodiments, i.e., where the order of steps is (a), (c),and (b), (b) is followed by: (d) contacting the textile with theperhydrolase enzyme system to perform a third color modification of thedyed textile.

In some embodiments, (a) is preceded by an enzymatic desizing step,which may be performed in the same bath as (a). In some embodiments, (c)is followed by the addition of a catalase enzyme. In some embodiments,catalase enzyme is added to the same bath in which any of (a), (b),and/or (c) are performed.

Regarding either of the aforementioned aspects, in some embodiments, thecellulase is an acid cellulase. In some embodiments, the cellulase is aneutral cellulase. In some embodiments, the cellulase is an alkalinecellulase. In some embodiments, the cellulase is a combination ofcellulases.

In some embodiments of any of the aforementioned aspects, theperhydrolase enzyme system may comprise a perhydrolase enzyme and anester substrate, wherein the perhydrolase enzyme catalyzes perhydrolysisof the ester substrate with a perhydrolysis:hydrolysis ratio equal to orgreater than 1. In some embodiments, the perhydrolase enzyme systemcomprises a Mycobacterium smegmatis perhydrolase or a variant, thereof.In some embodiments, the perhydrolase enzyme is a S54V variant ofMycobacterium smegmatis perhydrolase, or a variant, thereof.

In some embodiments, the laccase enzyme may be a Cerrena unicolorlaccase, or a variant, thereof.

In some embodiments, the textile is denim. In some embodiments, the dyeis indigo dye. In some embodiments, the dye is sulfur dye.

In another aspect, a textile produced by any of the preceding methods isprovided. In particular embodiments, the textile is indigo-dyed denim.In particular embodiments, the textile is sulfur-dyed denim.

In another aspect, a kit of parts for performing the foregoing methodsis provided.

These and other aspects and embodiments of present compositions andmethods will be further apparent from the description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table showing exemplary finishes and colors that can beobtained with cone denim XMISP using various embodiments of the presentcompositions and methods.

FIG. 2 is a table showing exemplary finishes and colors that can beobtained with cone denim 4671P using various embodiments of the presentcompositions and methods.

FIG. 3 is a table showing exemplary finishes and colors that can beobtained with cone denim 8349P using various embodiments of the presentcompositions and methods.

FIG. 4 is a table showing exemplary finishes and colors that can beobtained with cone denim W333 using various embodiments of the presentcompositions and methods.

FIG. 5 is a table showing exemplary finishes and colors that can beobtained with cone denim XOBBP using various embodiments of the presentcompositions and methods.

DETAILED DESCRIPTION Overview

Described are enzymatic compositions and methods for combined textileabrading and color-modification. In some embodiments, the combinedabrading and color modification are performed in a single bath, withoutthe need to rinse the textiles between processing steps. In someembodiments, abrading can be combined with color modification usingdifferent enzyme systems, such as perhydrolase enzyme system and alaccase enzyme system, to produce a wide range of finishes and colors.In the case of indigo and/or sulfur-dyed denim, i.e., textiles subjectedto a wide range of different chemical and physical treatments in pursuitof fashion, the present compositions and methods offer a comprehensiveenzymatic solution for obtaining known finishes and colors, and makepossible new finishes and colors.

Combined with previously-described enzymatic desizing and bleachclean-up methods, the present compositions and methods further fulfillthe need for start-to-finish enzymatic textile processing solutions thatare cost effective, environmentally friendly, and sufficiently versatileto produce a wide range of finishes and colors. These and other featuresand advantages of the present compositions and methods are furtherdescribed, herein.

Definitions

Prior to describing the present compositions and methods in detail, thefollowing terms are defined for clarity. Terms not defined should begiven their ordinary meanings as using in the relevant art.

As used herein, a “perhydrolase” is an enzyme capable of catalyzing aperhydrolysis reaction that results in the production of a sufficientlyhigh amount of peracid for use in an oxidative dye decolorization methodas described. Generally, the perhydrolase enzyme exhibits a highperhydrolysis to hydrolysis ratio. In some embodiments, the perhydrolasecomprises, consists of, or consists essentially of the Mycobacteriumsmegmatis perhydrolase amino acid sequence set forth in SEQ ID NO: 1, ora variant or homolog thereof. In some embodiments, the perhydrolaseenzyme comprises acyltransferase and/or arylesterase activity.

As used herein, the terms “perhydrolyzation,” “perhydrolyze,” or“perhydrolysis” refer to a reaction wherein a peracid is generated fromester and hydrogen peroxide substrate. In some embodiments, theperhydrolyzation reaction is catalyzed with a perhydrolase, e.g., acyltransferase or aryl esterase, enzyme. In some embodiments, a peracid isproduced by perhydrolysis of an ester substrate of the formulaR₁C(═O)OR₂, where R₁ and R₂ are the same or different organic moieties,in the presence of hydrogen peroxide (H₂O₂). In some embodiments, —OR₂is —OH. In some embodiments, —OR₂ is replaced by —NH₂. In someembodiments, a peracid is produced by perhydrolysis of a carboxylic acidor amide substrate.

As used herein, an “effective amount of perhydrolase enzyme” refers tothe quantity of perhydrolase enzyme necessary to produce thedecolorization effects described herein. Such effective amounts aredetermined by the skilled artisan in view of the present description,and are based on several factors, such as the particular enzyme variantused, the pH used, the temperature used, and the like, as well as theresults desired (e.g., level of whiteness).

As used herein, the term “peracid” refers to a molecule derived from acarboxylic acid ester that has been reacted with hydrogen peroxide toform a highly reactive product having the general formula RC(═O)OOH.Such peracid products are able to transfer one of their oxygen atoms toanother molecule, such as a dye. It is this ability to transfer oxygenatoms that enables a peracid, for example, peracetic acid, to functionas a bleaching agent.

As used herein, an “ester substrate,” with reference to an oxidative dyedecolorization system containing a perhydrolase enzyme, refers to aperhydrolase substrate that contains an ester linkage. Esters comprisingaliphatic and/or aromatic carboxylic acids and alcohols may be utilizedas substrates with perhydrolase enzymes. In some embodiments, the estersource is an acetate ester. In some embodiments, the ester source isselected from one or more of propylene glycol diacetate, ethylene glycoldiacetate, triacetin, ethyl acetate and tributyrin. In some embodiments,the ester source is selected from the esters of one or more of thefollowing acids: formic acid, acetic acid, propionic acid, butyric acid,valeric acid, caproic acid, caprylic acid, nonanoic acid, decanoic acid,dodecanoic acid, myristic acid, palmitic acid, stearic acid, and oleicacid.

As used herein, the term “hydrogen peroxide source” refers to a moleculecapable of generating hydrogen peroxide, e.g., in situ. Hydrogenperoxide sources include hydrogen peroxide, itself, as well as moleculesthat spontaneously or enzymatically produce hydrogen peroxide as areaction product. Such molecules include, e.g., perborate andpercarbonate.

As used herein, the phrase “perhydrolysis to hydrolysis ratio” refers tothe ratio of enzymatically produced peracid to enzymatically producedacid (e.g., in moles) that is produced by a perhydrolase enzyme from anester substrate under defined conditions and within a defined time. Insome embodiments, the assays provided in WO 05/056782 are used todetermine the amounts of peracid and acid produced by the enzyme.

As used herein, the term “acyl” refers to an organic group with thegeneral formula RCO—, derived from an organic acid by removal of the —OHgroup. Typically, acyl group names end with the suffix “-oyl,” e.g.,methanoyl chloride, CH₃CO—Cl, is the acyl chloride formed from methanoicacid, CH₃CO—OH).

As used herein, the term “acylation” refers to a chemical transformationin which one of the substituents of a molecule is substituted by an acylgroup, or the process of introduction of an acyl group into a molecule.

As used herein, the term “transferase” refers to an enzyme thatcatalyzes the transfer of a functional group from one substrate toanother substrate. For example, an acyl transferase may transfer an acylgroup from an ester substrate to a hydrogen peroxide substrate to form aperacid.

As used herein, the term “hydrogen peroxide generating oxidase” refersto an enzyme that catalyzes an oxidation/reduction reaction involvingmolecular oxygen (O₂) as the electron acceptor. In such a reaction,oxygen is reduced to water (H₂O) or hydrogen peroxide (H₂O₂). An oxidasesuitable for use herein is an oxidase that generates hydrogen peroxide(as opposed to water) on its substrate. An example of a hydrogenperoxide generating oxidase and its substrate suitable for use herein isglucose oxidase and glucose. Other oxidase enzymes that may be used forgeneration of hydrogen peroxide include alcohol oxidase, ethylene glycoloxidase, glycerol oxidase, amino acid oxidase, etc. In some embodiments,the hydrogen peroxide generating oxidase is a carbohydrate oxidase.

As used herein, a “laccase” is a multi-copper containing oxidase (EC1.10.3.2) that catalyzes the oxidation of phenols, polyphenols, andanilines by single-electron abstraction, with the concomitant reductionof oxygen to water in a four-electron transfer process.

As used herein, the term “textile” refers to fibers, yarns, fabrics,garments, and non-wovens. The term encompasses textiles made fromnatural, synthetic (e.g., manufactured), and various natural andsynthetic blends. Textiles may be unprocessed or processed fibers,yarns, woven or knit fabrics, non-wovens, and garments and may be madeusing a variety of materials, some of which are mentioned, herein.

As used herein, a “cellulosic” fiber, yarn or fabric is made at least inpart from cellulose. Examples include cotton and non-cotton cellulosicfibers, yarns or fabrics. Cellulosic fibers may optionally includenon-cellulosic fibers.

As used herein, a “non-cotton cellulosic” fiber, yarn or fabric iscomprised primarily of a cellulose based composition other than cotton.Examples include linen, ramie, jute, flax, rayon, lyocell, celluloseacetate, bamboo and other similar compositions, which are derived fromnon-cotton cellulosics.

As used herein, a “non-cellulosic” fiber, yarn or fabric is comprisedprimarily of a material other than cellulose. Examples includepolyester, nylon, rayon, acetate, lyocell, and the like.

As used herein, the term “fabric” refers to a manufactured assembly offibers and/or yarns that has substantial surface area in relation to itsthickness and sufficient cohesion to give the assembly useful mechanicalstrength.

As used herein, the term “dyeing,” refers to applying a color,especially by soaking in a coloring solution, to, for example, textiles.

As used herein, the term “dye” refers to a colored substance (i.e.,chromophore) that has an affinity to a substrate to which it is applied.Numerous classes of dyes are described herein.

As used herein, the terms “color modification” and “color adjustment”are used without distinction to refer to any change to the color of adyed textile resulting from the destruction, modification, or removal ofa dye associated with the textile. In some embodiments, the colormodification is decolorization (see below). Examples of colormodification include but are not limited to, bleaching, fading,imparting a grey cast, altering hue, saturation, or luminescence, andthe like. The amount and type of color modification can be determined bycomparing the color of a textile following enzymatic treatment with aperhydrolase enzyme (i.e., residual color) to the color of the textileprior to enzymatic treatment (i.e., original color) using knownspectrophotometric or visual inspection methods.

As used herein, the terms “decolorizing” and “decolorization” refer tocolor elimination or reduction via the destruction, modification, orremoval of dye, e.g., from an aqueous medium. In some embodiments,decolorizing or decolorization is defined as a percentage of colorremoval from aqueous medium. The amount of color removal can bedetermined by comparing the color of a textile following enzymatictreatment with a perhydrolase enzyme (i.e., residual color) to the colorof the textile prior to enzymatic treatment (i.e., original color) usingknown spectrophotometric or visual inspection methods.

As used herein, the term “original color” refers to the color of a dyedtextile prior to enzymatic treatment. Original color may be measuredusing known spectrophotometric or visual inspection methods.

As used herein, the term “residual color” refers to the color of a dyedtextile prior to enzymatic treatment. Residual color may be measuredusing known spectrophotometric or visual inspection methods.

As used herein, the terms “size” or “sizing” refer to compounds used inthe textile industry to improve weaving performance by increasing theabrasion resistance and strength of the yarn. Size is usually made of,for example, starch or starch-like compounds.

As used herein, the terms “desize” or “desizing” refer to the process ofeliminating size, generally starch, from textiles usually prior toapplying special finishes, dyes or bleaches.

As used herein a “desizing enzyme” is an enzyme used to remove size.Exemplary enzymes are amylases and mannanases.

As used herein, a “cellulase” is an enzyme capable of hydrolizingcellulose.

As used herein, an “acid cellulase” is a cellulase having a pH optima inthe acidic pH range, for example, from about pH 4.0 to about pH 5.5.

As used herein, a “neutral cellulase” is a cellulase having a pH optimain the neutral pH range, for example, from about pH 5.5 to about pH 7.5.

As used herein, an “alkaline cellulase” is a cellulase having a pHoptima in the alkaline pH range, for example, from about pH 7.5 to aboutpH 11.

As used herein, the term “abrading” refers generally to contacting atextile comprising cellulose fibers with one or more cellulases toproduce an effect. Such effects include but are not limited tosoftening, smoothing, defuzzing, depilling, biopolishing, and/orintentionally distressing the textile, locally or in its entirety. Insome cases, more than one abrading step may be desirable.

As used herein, an “aqueous medium” is a solution and/or suspensionprimarily comprising water as a solvent. The aqueous medium typicallyincludes at least one dye to be decolorized, as well as any number ofdissolved or suspended components, including but not limited tosurfactants, salts, buffers, stabilizers, complexing agents, chelatingagents, builders, metal ions, additional enzymes and substrates, and thelike. Exemplary aqueous media are textile dying solutions. Materialssuch as textile articles, textile fibers, and other solid materials mayalso be present in or in contact with the aqueous medium.

As used herein, the term “contacting,” means bringing into physicalcontact, such as by incubating a subject item (e.g., a textile) in thepresence of an aqueous solution containing a reaction component (e.g.,an enzyme).

As used herein, the term “sequential,” with reference to a plurality ofenzymatic treatments of a textile, means that a second specifiedenzymatic treatment is performed after a first specified enzymatictreatment is performed. Sequential treatments may be separated byintervening wash steps. Where specified, sequential enzymatic treatmentsmay be performed “in the same bath,” meaning in the substantially thesame liquid medium without intervening wash steps. Single-bathsequential treatment may include pH adjustments, temperature adjustment,and/or the addition of salts, activators, mediators, and the like, butshould not include washes, rinses, or “dropping the bath” between firstand second enzymatic treatments.

As used herein, the term “simultaneous,” with reference to a pluralityof enzymatic treatments of a textile, means that a second specifiedenzymatic treatment is performed at the same time (i.e., at leastpartially overlapping with) as a first specified enzymatic treatment.Simultaneous enzymatic treatments are necessarily performed “in the samebath” without intervening wash steps.

As used herein, “packaging” refers to a container capable of providing aperhydrolase enzyme, substrate for the perhydrolase enzyme, and/orhydrogen peroxide source in an easy to handle and transport form.Exemplary packaging includes boxes, tubs, cans, barrels, drums, bags, oreven tanker trucks.

As used herein, the terms “purified” and “isolated” refer to the removalof contaminants from a sample and/or to a material (e.g., a protein,nucleic acid, cell, etc.) that is removed from at least one componentwith which it is naturally associated. For example, these terms mayrefer to a material which is substantially or essentially free fromcomponents which normally accompany it as found in its native state,such as, for example, an intact biological system

As used herein, the term “polynucleotide” refers to a polymeric form ofnucleotides of any length and any three-dimensional structure andsingle- or multi-stranded (e.g., single-stranded, double-stranded,triple-helical, etc.), which contain deoxyribonucleotides,ribonucleotides, and/or analogs or modified forms ofdeoxyribonucleotides or ribonucleotides, including modified nucleotidesor bases or their analogs. Because the genetic code is degenerate, morethan one codon may be used to encode a particular amino acid. Any typeof modified nucleotide or nucleotide analog may be used, so long as thepolynucleotide retains the desired functionality under conditions ofuse, including modifications that increase nuclease resistance (e.g.,deoxy, 2′-O-Me, phosphorothioates, etc.). Labels may also beincorporated for purposes of detection or capture, for example,radioactive or nonradioactive labels or anchors, e.g., biotin. The termpolynucleotide also includes peptide nucleic acids (PNA).Polynucleotides may be naturally occurring or non-naturally occurring.The terms “polynucleotide” and “nucleic acid” and “oligonucleotide” areused herein interchangeably. Polynucleotides may contain RNA, DNA, orboth, and/or modified forms and/or analogs thereof. A sequence ofnucleotides may be interrupted by non-nucleotide components. One or morephosphodiester linkages may be replaced by alternative linking groups.These alternative linking groups include, but are not limited to,embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S(“dithioate”), (O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. Polynucleotides may belinear or circular or comprise a combination of linear and circularportions.

As used herein, “polypeptide” refers to any composition comprised ofamino acids and recognized as a protein by those of skill in the art.The conventional one-letter or three-letter code for amino acid residuesis used herein. The terms “polypeptide” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art.

As used herein, functionally and/or structurally similar proteins areconsidered to be “related proteins.” In some embodiments, these proteinsare derived from a different genus and/or species, including differencesbetween classes of organisms (e.g., a bacterial protein and a fungalprotein). In additional embodiments, related proteins are provided fromthe same species. Indeed, it is not intended that the processes, methodsand/or compositions described herein be limited to related proteins fromany particular source(s). In addition, the term “related proteins”encompasses tertiary structural homologs and primary sequence homologs.In further embodiments, the term encompasses proteins that areimmunologically cross-reactive.

As used herein, the term “derivative” refers to a protein which isderived from a protein by addition of one or more amino acids to eitheror both the C- and N-terminal end(s), substitution of one or more aminoacids at one or a number of different sites in the amino acid sequence,and/or deletion of one or more amino acids at either or both ends of theprotein or at one or more sites in the amino acid sequence, and/orinsertion of one or more amino acids at one or more sites in the aminoacid sequence. The preparation of a protein derivative is preferablyachieved by modifying a DNA sequence which encodes for the nativeprotein, transformation of that DNA sequence into a suitable host, andexpression of the modified DNA sequence to form the derivative protein.

Related (and derivative) proteins comprise “variant proteins.” In someembodiments, variant proteins differ from a parent protein, e.g., awild-type protein, and one another by a small number of amino acidresidues. The number of differing amino acid residues may be one ormore, for example, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, or more aminoacid residues. In some aspects, related proteins and particularlyvariant proteins comprise at least 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even99% or more amino acid sequence identity. Additionally, a relatedprotein or a variant protein refers to a protein that differs fromanother related protein or a parent protein in the number of prominentregions. For example, in some embodiments, variant proteins have 1, 2,3, 4, 5, or 10 corresponding prominent regions that differ from theparent protein. Prominent regions include structural features, conservedregions, epitopes, domains, motifs, and the like.

Methods are known in the art that are suitable for generating variantsof the enzymes described herein, including but not limited tosite-saturation mutagenesis, scanning mutagenesis, insertionalmutagenesis, random mutagenesis, site-directed mutagenesis, anddirected-evolution, as well as various other recombinatorial approaches.Note that where a particular mutation in a variant polypeptide isspecified, further variants of that variant polypeptide retain thespecified mutation and vary at other positions not specified.

As used herein, the term “analogous sequence” refers to a sequencewithin a protein that provides similar function, tertiary structure,and/or conserved residues as the protein of interest (i.e., typicallythe original protein of interest). For example, in epitope regions thatcontain an alpha-helix or a beta-sheet structure, the replacement aminoacids in the analogous sequence preferably maintain the same specificstructure. The term also refers to nucleotide sequences, as well asamino acid sequences. In some embodiments, analogous sequences aredeveloped such that the replacement amino acids result in a variantenzyme showing a similar or improved function. In some embodiments, thetertiary structure and/or conserved residues of the amino acids in theprotein of interest are located at or near the segment or fragment ofinterest. Thus, where the segment or fragment of interest contains, forexample, an alpha-helix or a beta-sheet structure, the replacement aminoacids preferably maintain that specific structure.

As used herein, the term “homologous protein” refers to a protein thathas similar activity and/or structure to a reference protein. It is notintended that homologs necessarily be evolutionarily related. Thus, itis intended that the term encompass the same, similar, or correspondingenzyme(s) (i.e., in terms of structure and function) obtained fromdifferent organisms. In some embodiments, it is desirable to identify ahomolog that has a quaternary, tertiary and/or primary structure similarto the reference protein. In some embodiments, homologous proteinsinduce similar immunological response(s) as a reference protein. In someembodiments, homologous proteins are engineered to produce enzymes withdesired activity(ies).

The degree of homology between sequences may be determined using anysuitable method known in the art (see, e.g., Smith and Waterman (1981)Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol.,48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444;programs such as GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package (Genetics Computer Group, Madison, Wis.); andDevereux et al. (1984) Nucleic Acids Res. 12:387-395).

For example, PILEUP is a useful program to determine sequence homologylevels. PILEUP creates a multiple sequence alignment from a group ofrelated sequences using progressive, pair-wise alignments. It can alsoplot a tree showing the clustering relationships used to create thealignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng and Doolittle, (Feng and Doolittle (1987) J. Mol. Evol.35:351-360). The method is similar to that described by Higgins andSharp (Higgins and Sharp (1989) CABIOS 5:151-153). Useful PILEUPparameters including a default gap weight of 3.00, a default gap lengthweight of 0.10, and weighted end gaps. Another example of a usefulalgorithm is the BLAST algorithm, described by Altschul et al. (Altschulet al. (1990) J. Mol. Biol. 215:403-410; and Karlin et al. (1993) Proc.Natl. Acad. Sci. USA 90:5873-5787). One particularly useful BLASTprogram is the WU-BLAST-2 program (See, Altschul et al. (1996) Meth.Enzymol. 266:460-480). Parameters “W,” “T,” and “X” determine thesensitivity and speed of the alignment. The BLAST program uses asdefaults a word-length (W) of 11, the BLOSUM62 scoring matrix (See,Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M′5, N′-4, and a comparisonof both strands.

As used herein, the phrases “substantially similar” and “substantiallyidentical,” in the context of at least two nucleic acids orpolypeptides, typically means that a polynucleotide or polypeptidecomprises a sequence that has at least about 40% identity, morepreferable at least about 50% identity, yet more preferably at leastabout 60% identity, preferably at least about 75% identity, morepreferably at least about 80% identity, yet more preferably at leastabout 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, or even at least about 99% sequenceidentity, compared to the reference (i.e., wild-type) sequence. Sequenceidentity may be determined using known programs such as BLAST, ALIGN,and CLUSTAL using standard parameters. (See e.g., Altschul, et al.(1990) J. Mol. Biol. 215:403-410; Henikoff et al. (1989) Proc. Natl.Acad. Sci. USA 89:10915; Karin et al. (1993) Proc. Natl. Acad. Sci. USA90:5873; and Higgins et al. (1988) Gene 73:237-244). Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. Also, databases may be searchedusing FASTA (Pearson et al. (1988) Proc. Natl. Acad. Sci. USA85:2444-2448). One indication that two polypeptides are substantiallyidentical is that the first polypeptide is immunologicallycross-reactive with the second polypeptide. Typically, polypeptides thatdiffer by conservative amino acid substitutions are immunologicallycross-reactive. Thus, a polypeptide is substantially identical to asecond polypeptide, for example, where the two peptides differ only by aconservative substitution. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions (e.g., within a rangeof medium to high stringency).

As used herein, “wild-type” and “native” proteins are those found innature. The terms “wild-type sequence,” and “wild-type gene” are usedinterchangeably herein, to refer to a sequence that is native ornaturally occurring in a host cell. In some embodiments, the wild-typesequence refers to a sequence of interest that is the starting point ofa protein engineering project. The genes encoding thenaturally-occurring protein may be obtained in accord with the generalmethods known to those skilled in the art. The methods generallycomprise synthesizing labeled probes having putative sequences encodingregions of the protein of interest, preparing genomic libraries fromorganisms expressing the protein, and screening the libraries for thegene of interest by hybridization to the probes. Positively hybridizingclones are then mapped and sequenced.

As used herein, the singular articles “a,” “an,” and “the” encompass theplural referents unless the context clearly dictates otherwise. Allreferences sited herein are hereby incorporated by reference in theirentirety.

The following abbreviations/acronyms have the following meanings unlessotherwise specified:

-   -   cDNA complementary DNA    -   DNA deoxyribonucleic acid    -   EC enzyme commission    -   kDa kiloDalton    -   MW molecular weight    -   SDS-PAGE sodium dodecyl sulfate polyacrylamide gel        electrophoresis    -   w/v weight/volume    -   w/w weight/weight    -   v/v volume/volume    -   wt % weight percent    -   ° C. degrees Centigrade    -   H₂O water    -   H₂O₂ hydrogen peroxide    -   dH₂O or DI deionized water    -   dIH₂O deionized water, Milli-Q filtration    -   g or gm gram    -   μ microgram    -   mg milligram    -   kg kilogram    -   μL and μl microliter    -   mL and ml milliliter    -   mm millimeter    -   μm micrometer    -   M molar    -   mM millimolar    -   μM micromolar    -   U unit    -   ppm parts per million    -   sec and ″ second    -   min and ′ minute    -   hr hour    -   ETOH ethanol    -   eq. equivalent    -   N normal    -   CI Colour (Color) Index    -   CAS Chemical Abstracts Society

Cellulases

In some embodiments, color modification is performed sequentially orsimultaneously in the same bath as abrading using one or more cellulaseenzymes. Cellulases are typically used prior to, or concurrent with,treatment with a perhydrolase system or laccase system. In someembodiments, a plurality of cellulases may be used together orseparately in different steps.

Cellulases are classified in enzyme families encompassing endo- andexo-activities as well as cellobiose hydrolyzing capability. Cellulasesare also characterized as acid cellulases, neutral cellulases, oralkaline cellulases, based on their pH optima.

Cellulases may be derived from microorganisms which are known to becapable of producing cellulolytic enzymes, such as, e.g., species ofTrichoderma, Humicola, Fusarium, Aspergillus, Thermomyces, Bacillus,Myceliophthora, Phanerochaete, Irpex, Scytalidium, Schizophyllum,Penicillium, Geotricum, and Staphylotrichum. Known species capable forproducing celluloytic enzymes include Humicola insolens, Fusariumoxysporum or Trichoderma reesei. Exemplary cellulases include theendoglucanase from Streptomyces sp. 11AG8, the neutral cellulases fromStaphylotrichum coccosporum and Humicola insolens, and individualcellulases and cellulase blends from T. reesei.

Non-limiting examples of suitable cellulases are disclosed in U.S. Pat.No. 4,435,307; European Patent Application Nos. EP 0 495 257 and EP 271004; and PCT Patent Application No. WO91/17244, WO92/06221, WO98/003667.WO01/090375, WO05/054475, and WO05/056787.

In some embodiments, the cellulase may be used in a concentration in therange from about 0.0001% to about 1% enzyme protein by weight of thefabric, such as about 0.0001% to about 0.05% enzyme protein by weight ofthe fabric, or about 0.0001 to about 0.01% enzyme protein by weight ofthe fabric.

The cellulolytic activity may be determined in endo-cellulase units(ECU) by measuring the ability of the enzyme to reduce the viscosity ofa solution of carboxymethyl cellulose (CMC), The ECU assay quantifiesthe amount of catalytic activity present in the sample by measuring theability of the sample to reduce the viscosity of a solution ofcarboxy-methylcellulose (CMC). The assay is carried out in a vibrationviscosimeter (e.g., MIVI 3000 from Sofraser, France) at 40° C.; pH 7.5;0.1 M phosphate buffer; time 30 minutes using a relative enzyme standardfor reducing the viscosity of the CHIC substrate (Hercules 7 LED),enzyme concentration approx. 0.15 ECU/ml. The arch standard is definedto 8200 ECU/g. One ECU is amount of enzyme that reduces the viscosity toone half under these conditions.

Perhydrolase Enzyme System

The present compositions and methods utilize a perhydrolase enzymesystem, comprising a perhydrolase enzyme capable of generating peracidsin the present of a suitable ester substrate and hydrogen peroxidesource.

In some embodiments, the perhydrolase enzyme is naturally-occurringenzyme. In some embodiments, a perhydrolase enzyme comprises, consistsof, or consists essentially of an amino acid sequence that is at leastabout 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or even 99.5% identical to the amino acid sequence of anaturally-occurring perhydrolase enzyme. In some embodiments, theperhydrolase enzyme is from a microbial source, such as a bacterium orfungus.

In some embodiments, the perhydrolase enzyme is a naturally occurringMycobacterium smegmatis perhydrolase enzyme or a variant thereof. Thisenzyme, its enzymatic properties, its structure, and numerous variantsand homologs, thereof, are described in detail in International PatentApplication Publications WO 05/056782A and WO 08/063,400A and U.S.Patent Application Publications US2008145353 and US2007167344, which areincorporated by reference.

In some embodiments, the perhydrolase enzyme has aperhydrolysis:hydrolysis ratio of at least 1. In some embodiments, theperhydrolase enzyme has a perhydrolysis:hydrolysis ratio greater than 1.In some embodiments, the perhydrolysis:hydrolysis ratio is greater than1.5, greater than 2.0, greater than 2.5, or even greater than 3.0. Thesehigh perhydrolysis:hydrolysis ratios are features unique to of M.smegmatis perhydrolase and variants, thereof.

The amino acid sequence of M. smegmatis perhydrolase is shown below (SEQID NO. 1):

MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIEEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDTKAYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPHPWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFTEAN NRDLGVALAEQVRSLL

The corresponding polynucleotide sequence encoding M. smegmatisperhydrolase is shown below (SEQ ID NO: 2):

5′-ATGGCCAAGCGAATTCTGTGTTTCGGTGATTCCCTGACCTGGGGCTGGGTCCCCGTCGAAGACGGGGCACCCACCGAGCGGTTCGCCCCCGACGTGCGCTGGACCGGTGTGCTGGCCCAGCAGCTCGGAGCGGACTTCGAGGTGATCGAGGAGGGACTGAGCGCGCGCACCACCAACATCGACGACCCCACCGATCCGCGGCTCAACGGCGCGAGCTACCTGCCGTCGTGCCTCGCGACGCACCTGCCGCTCGACCTGGTGATCATCATGCTGGGCACCAACGACACCAAGGCCTACTTCCGGCGCACCCCGCTCGACATCGCGCTGGGCATGTCGGTGCTCGTCACGCAGGTGCTCACCAGCGCGGGCGGCGTCGGCACCACGTACCCGGCACCCAAGGTGCTGGTGGTCTCGCCGCCACCGCTGGCGCCCATGCCGCACCCCTGGTTCCAGTTGATCTTCGAGGGCGGCGAGCAGAAGACCACTGAGCTCGCCCGCGTGTACAGCGCGCTCGCGTCGTTCATGAAGGTGCCGTTCTTCGACGCGGGTTCGGTGATCAGCACCGACGGCGTCGACGGAATCCACTTCACCGAGGCCAACAATCGCGATCTCGGGGTGGCCCTCGCGGAACAGGTGCGGAGCCTGCT GTAA-3′

In some embodiments, a perhydrolase enzyme comprises, consists of, orconsists essentially of the amino acid sequence set forth in SEQ ID NO:1 or a variant or homologue thereof. In some embodiments, theperhydrolase enzyme comprises, consists of, or consists essentially ofan amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% identical tothe amino acid sequence set forth in SEQ ID NO: 1.

In some embodiments, the perhydrolase enzyme comprises one or moresubstitutions at one or more amino acid positions equivalent toposition(s) in the M. smegmatis perhydrolase amino acid sequence setforth in SEQ ID NO: 1. In some embodiments, the perhydrolase enzymecomprises any one or any combination of substitutions of amino acidsselected from M1, K3, R4, I5, L6, C7, D10, S11, L12, T13, W14, W16, G15,V17, P18, V19, D21, G22, A23, P24, T25, E26, R27, F28, A29, P30, D31,V32, R33, W34, T35, G36, L38, Q40, Q41, D45, L42, G43, A44, F46, E47,V48, I49, E50, E51, G52, L53, S54, A55, R56, T57, T58, N59, I60, D61,D62, P63, T64, D65, P66, R67, L68, N69, G70, A71, S72, Y73, S76, C77,L78, A79, T80, L82, P83, L84, D85, L86, V87, N94, D95, T96, K97,Y99F100, R101, R102, P104, L105, D106, I107, A108, L109, G110, M111,S112, V113, L114, V115, T116, Q117, V118, L119, T120, S121, A122, G124,V125, G126, T127, T128, Y129, P146, P148, W149, F150, I153, F154, I194,and F196.

In some embodiments, the perhydrolase enzyme comprises one or more ofthe following substitutions at one or more amino acid positionsequivalent to position(s) in the M. smegmatis perhydrolase amino acidsequence set forth in SEQ ID NO: 1: L12C, Q, or G; T25S, G, or P; L53H,Q, G, or S; S54V, L A, P, T, or R; A55G or T; R67T, Q, N, G, E, L, or F;K97R; V125S, G, R, A, or P; F154Y; F196G.

In some embodiments, the perhydrolase enzyme comprises a combination ofamino acid substitutions at amino acid positions equivalent to aminoacid positions in the M. smegmatis perhydrolase amino acid sequence setforth in SEQ ID NO: 1: L12I S54V; L12M S54T; L12T S54V; L12Q T25S S54V;L53H S54V; S54P V125R; S54V V125G; S54V F196G; S54V K97R V125G; or A55GR67T K97R V125G.

In particular embodiments, the perhydrolase enzyme is the S54V variantof the M. smegmatis perhydrolase, which is shown, below (SEQ ID NO: 3);S54V substitution underlined):

MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIEEGLVARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDTKAYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPHPWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFTEAN NRDLGVALAEQVRSLL

In some embodiments, the perhydrolase enzyme includes the S54Vsubstitution but is otherwise at least about 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% identical tothe amino acid sequence set forth in SEQ ID NOs: 1 or 3.

In some embodiments, the perhydrolase enzyme is provided at aconcentration of about 1 to about 100 ppm, or more. In some embodiments,the perhydrolase enzyme is provided at a molar ratio with respect to theamount of dye on the textile. In some embodiments, the molar ratio isfrom about 1/10,000 to about 1/10, or even from about ⅕,000 to about1/100. In some embodiments, the concentration of perhydrolase enzyme isfrom about 10⁻⁹ M to about 10⁻⁵ M, from about 10⁻⁸ M to about 10⁻⁵ M,from about 10⁻⁸ M to about 10⁻⁶ M, about 5×10⁻⁸ M to about 5×10⁻⁷ M, oreven about 10⁻⁷ M to about 5×10⁻⁷ M. In some embodiments, the amount ofperhydrolase enzyme is below a predetermined amount to improve theefficiency of color modification.

The perhydrolase enzyme system may include at least one ester moleculethat serves as a substrate for the perhydrolase enzyme for production ofa peracid in the presence of hydrogen peroxide. In some embodiments, theester substrate is an ester of an aliphatic and/or aromatic carboxylicacid or alcohol. The ester substrate may be a mono-, di-, or multivalentester, or a mixture thereof. For example, the ester substrate may be acarboxylic acid and a single alcohol (monovalent, e.g., ethyl acetate,propyl acetate), two carboxylic acids and a diol [e.g., propylene glycoldiacetate (PGDA), ethylene glycol diacetate (EGDA), or a mixture, forexample, 2-acetyloxy 1-propionate, where propylene glycol has an acetateester on alcohol group 2 and a propyl ester on alcohol group 1], orthree carboxylic acids and a triol (e.g., glycerol triacetate or amixture of acetate/propionate, etc., attached to glycerol or anothermultivalent alcohol).

In some embodiments, the ester substrate is an ester of a nitroalcohol(e.g., 2-nitro-1-propanol). In some embodiments, the ester substrate isa polymeric ester, for example, a partially acylated (acetylated,propionylated, etc.) poly carboxy alcohol, acetylated starch, etc. Insome embodiments, the ester substrate is an ester of one or more of thefollowing: formic acid, acetic acid, propionic acid, butyric acid,valeric acid, caproic acid, caprylic acid, nonanoic acid, decanoic acid,dodecanoic acid, myristic acid, palmitic acid, stearic acid, and oleicacid. In some embodiments, triacetin, tributyrin, and other esters serveas acyl donors for peracid formation. In some embodiments, the estersubstrate is propylene glycol diacetate, ethylene glycol diacetate, orethyl acetate. In one embodiment, the ester substrate is propyleneglycol diacetate.

As noted above, suitable substrates may be monovalent (i.e., comprisinga single carboxylic acid ester moiety) or plurivalent (i.e., comprisingmore than one carboxylic acid ester moiety). The amount of substrateused for color modification may be adjusted depending on the numbercarboxylic acid ester moieties in the substrate molecule. In someembodiments, the concentration of carboxylic acid ester moieties in theaqueous medium is about 20-500 mM, for example, about 40 mM to about 400mM, about 40 mM to about 200 mM, or even about 60 mM to about 200 mM.Exemplary concentrations of carboxylic acid ester moieties include about60 mM, about 80 mM, about 100 mM, about 120 mM, about 140 mM, about 160mM, about 180 mM, and about 200 mM.

In some embodiments, where the ester substrate is divalent (as in thecase of EGDA) it is provided in an amount of about 10-200 mM, forexample, about 20 mM to about 200 mM, about 20 mM to about 100 mM, oreven about 30 mM to about 100 mM. Exemplary amounts of ester substrateinclude about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM,about 80 mM, about 90 mM, and about 100 mM. The skilled person canreadily calculate the corresponding amounts of trivalent, or otherplurivalent ester substrates based on the number of carboxylic acidesters moieties per molecule.

In some embodiments, the ester substrate is provided in a molar excesswith respect to the molar amount of dye on the textile to be subjectedto color modification. In some embodiments, the carboxylic acid estermoieties of the ester substrate are provided at about 20 to about 20,000times the molar amount of dye. Exemplary molar ratios of carboxylic acidester moieties to dye molecules are from about 100/1 to about 10,000/1,from about 1,000/1 to about 10,000/1, or even 2,000/1 to about 6,000/1.In some cases, the molar ratio of ester substrate to dye molecules is atleast 2,000/1, or at least 6,000/1.

In some embodiments, where the ester substrate is divalent (as in thecase of EGDA) the ester substrate is provided at about 10 to about10,000 times the molar amount of dye. Exemplary molar ratios of estersubstrate to dye molecules are from about 50/1 to about 5,000/1, fromabout 500/1 to about 5,000/1, or even 1,000/1 to about 3,000/1. In somecases, the molar ratio of ester substrate to dye molecules is at least1,000/1, or at least 3,000/1. As before, the skilled person can readilycalculate the corresponding amounts of trivalent, or other plurivalentester substrates based on the number of carboxylic acid esters moietiesper molecule.

In some embodiments, the ester substrate is provided at a concentrationof about 100 ppm to about 100,000 ppm, ppm, or about 2500 to about 3500ppm. In some embodiments, the ester substrate is provided in a molarexcess with respect to the perhydrolase enzyme. In some embodiments, themolar ratio of carboxylic acid ester moieties to perhydrolase enzyme isat least about 2×10⁵/1, at least about 4×10⁵/1, at least about 1×10⁶/1,at least about 2×10⁶/1, at least about 4×10⁶/1, or even at least about1×10⁷/1, or more. In some embodiments, the ester substrate is providedin a molar excess of from about 4×10⁵/1, to about 4×10⁶/1, with respectto the perhydrolase enzyme.

In some embodiments, where the ester substrate is divalent (as in thecase of EGDA), the molar ratio of ester substrate to perhydrolase enzymeis at least about 1×10⁵/1, at least about 2×10⁵/1, at least about5×10⁵/1, at least about 1×10⁶/1, at least about 2×10⁶/1, or even atleast about 5×10⁶/1, or more. In some embodiments, the ester substrateis provided in a molar excess of from about 2×10⁵/1 to about 2×10⁶/1,with respect to the perhydrolase enzyme. The skilled person can readilycalculate the corresponding amounts of trivalent, or other plurivalentester substrates based on the number of carboxylic acid esters moietiesper molecule.

The perhydrolase enzyme system further includes at least one hydrogenperoxide source. Generally, hydrogen peroxide can be provided directly(i.e., in batch), or generated continuously (i.e., in situ) by chemical,electro-chemical, and/or enzymatic means.

In some embodiments, the hydrogen peroxide source is hydrogen peroxide,itself. In some embodiments, the hydrogen peroxide source is a compoundthat generates hydrogen peroxide upon addition to water. The compoundmay be a solid compound. Such compounds include adducts of hydrogenperoxide with various inorganic or organic compounds, of which the mostwidely employed is sodium carbonate per hydrate, also referred to assodium percarbonate.

In some embodiments, the hydrogen peroxide source is an inorganicperhydrate salt. Examples of inorganic perhydrate salts are perborate,percarbonate, perphosphate, persulfate and persilicate salts. Inorganicperhydrate salts are normally alkali metal salts. Additional hydrogenperoxide sources include adducts of hydrogen peroxide with zeolites, orurea hydrogen peroxide.

The hydrogen peroxide source may be in a crystalline form and/orsubstantially pure solid form without additional protection. For certainperhydrate salts, preferred forms are granular compositions involving acoating, which provides better storage stability for the perhydrate saltin the granular product. Suitable coatings comprise inorganic salts suchas alkali metal silicate, carbonate or borate salts or mixtures thereof,or organic materials such as waxes, oils, or fatty soaps.

In some embodiments, the hydrogen peroxide source is an enzymatichydrogen peroxide generation system. In one embodiment, the enzymatichydrogen peroxide generation system comprises an oxidase and itssubstrate. Suitable oxidase enzymes include, but are not limited to:glucose oxidase, sorbitol oxidase, hexose oxidase, choline oxidase,alcohol oxidase, glycerol oxidase, cholesterol oxidase, pyranoseoxidase, carboxyalcohol oxidase, L-amino acid oxidase, glycine oxidase,pyruvate oxidase, glutamate oxidase, sarcosine oxidase, lysine oxidase,lactate oxidase, vanillyl oxidase, glycolate oxidase, galactose oxidase,uricase, oxalate oxidase, and xanthine oxidase.

The following equation provides an example of a coupled system forenzymatic production of hydrogen peroxide.

It is not intended that the generation of H₂O₂ be limited to anyspecific enzyme, as any enzyme that generates H₂O₂ with a suitablesubstrate may be used. For example, lactate oxidases from Lactobacillusspecies known to create H₂O₂ from lactic acid and oxygen may be used.One advantage of such a reaction is the enzymatic generation of acid(e.g., gluconic acid in the above example), which reduces the pH of abasic aqueous solution to within the pH range in which peracid is mosteffective in bleaching (i.e., at or below the pKa). Such a reduction inpH is also brought about directly by the production of peracid. Otherenzymes (e.g., alcohol oxidase, ethylene glycol oxidase, glyceroloxidase, amino acid oxidase, etc.) that are capable of generatinghydrogen peroxide may also be used with ester substrates in combinationwith a perhydrolase enzyme to generate peracids.

Where hydrogen peroxide is generated electrochemically, it may beproduced, for example, using a fuel cell supplied with oxygen andhydrogen gas.

In some embodiments, hydrogen peroxide is provided at a concentration ofabout 100 ppm to about 10,000 ppm, about 1,000 ppm to about 3,000 ppm,or about 1,500 to about 2,500 ppm. In some embodiments, hydrogenperoxide is provided at about 10 to about 1,000 times the molar amountof dye.

In some embodiments, hydrogen peroxide is provided in an amount of about10-200 mM, for example, about 20 mM to about 200 mM, about 20 mM toabout 100 mM, or even about 30 mM to about 100 mM. Exemplary amounts ofhydrogen peroxide include about 30 mM, about 40 mM, about 50 mM, about60 mM, about 70 mM, about 80 mM, about 90 mM, and about 100 mM.

In some embodiments, hydrogen peroxide is provided in a molar excesswith respect to the molar amount of dye to be subjected to colormodification. In some embodiments, the hydrogen peroxide is provided atabout 10 to about 10,000 times the molar amount of dye. Exemplary molarratios of hydrogen peroxide to dye molecules are from about 500/1 toabout 5,000/1, or even 1,000/1 to about 3,000/1. In some cases, themolar ratio of hydrogen peroxide to dye molecules is at least 1,000/1,or at least 3,000/1.

In some embodiments, the hydrogen peroxide is provided in a molar excesswith respect to the perhydrolase enzyme. In some embodiments, the molarratio of hydrogen peroxide to perhydrolase enzyme is at least about1×10⁵/1, at least about 2×10⁵/1, at least about 5×10⁵/1, at least about1×10⁶/1, at least about 2×10⁶/1, or even at least about 5×10⁶/1, ormore. In some embodiments, the hydrogen peroxide is provided in a molarexcess of about 2×10⁵/1 to 2×10⁶/1, with respect to the perhydrolaseenzyme.

It may in some circumstances be desirable to add catalase to the textilebath to destroy residual hydrogen peroxide. In such cases, catalase cangenerally be added directly to the bath, without prior rinsing of thetextiles.

Laccase Enzyme System

In some embodiments, the compositions and methods include treatment witha laccase or related enzyme system to effect a cast, color, or shadechange of the textile. The laccase system may be used sequentially withtreatment with a perhydrolase enzyme. Moreover, the laccase system canbe used before or after the perhydrolase system to produce a wide rangeof finishes and colors.

Laccases and laccase-related enzymes include enzymes of theclassification EC 1.10.3.2. Laccase enzymes are known from microbial andplant origin. A microbial laccase enzyme may be derived from bacteria orfungi (including filamentous fungi and yeasts) and suitable examplesinclude a laccase derivable from a strain of Aspergillus, Neurospora,e.g., N. crassa. Podospora, Botrytis, Collybia, Cerrena, e.g., Cerrenaunicolor, Stachybotrys, Panus, e.g., Panus rudis, Thielavia, Fomes,Lentinus, Pleurotus, Trametes, e.g. T. villosa and T. versicolor,Rhizoctonia, e.g., R. solani, Coprinus, e.g. C. plicatilis and C.cinereus, Psatyrella, Myceliophthora, e.g., M. thermonhila,Schytalidium, Phlebia, e.g., P. radita (WO 92/01046), or Coriolus, e.g.,C. hirsutus (JP 2-238885), Spongipellis sp., Polyporus, Ceriporiopsissubvermispora, Ganoderma tsunodae and Trichoderma.

A laccase or laccase related enzyme may be produced by culturing a hostcell transformed with a recombinant DNA vector which includes a DNAsequence encoding the laccase as well as DNA sequences permitting theexpression of the DNA sequence encoding the laccase, in a culture mediumunder conditions permitting the expression of the laccase enzyme, andrecovering the laccase from the culture.

An expression vector containing a polynucleotide sequence encoding alaccase enzyme may be transformed into a suitable host cell. The hostcell may be a fungal cell, such as a filamentous fungal cell, examplesof which include but are not limited to species of Trichoderma (e.g.,Trichoderma reesei (previously classified as T. longibrachiatum andcurrently also known as Hypocrea jecorina), Trichoderma viride,Trichoderma koningii, Trichoderma harzianum), Aspergillus spp. (e.g.,Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Aspergillusawamori), Penicillium spp., Humicola spp. (e.g. Humicola insolens,Humicola grisea, Fusarium spp. (e.g., Fusarium graminum, Fusariumvenenatum), Neurospora spp., Hypocrea spp., and Mucor spp. A host cellfor expression of a laccase enzyme may also be a cell of a Cerrenaspecies, e.g., Cerrena unicolor. Fungal cells may be transformed by aprocess involving protoplast formation and transformation of theprotoplasts followed by regeneration of the cell wall using techniquesknown in the art. Alternatively, the host organism may be a bacterium,such as species of Bacillus spp. (e.g., Bacillus subtilis, Bacilluslicheniformis, Bacillus lentus, Bacillus stearothremophilus, Bacillusbrevis), Pseudomonas, Streptomyces (e.g., Streptomyces coelicolor,Streptomyces lividans), or E. coli. The transformation of bacterialcells may be performed according to conventional methods, e.g., asdescribed in T. Maniatis et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, 1982. The screening of appropriate DNA sequences andconstruction of vectors may also be carried out by standard procedures.

The medium used to culture the transformed host cells may be anyconventional medium suitable for growing the host cells. In someembodiments, the expressed enzyme is secreted into the culture mediumand may be recovered therefrom by well-known procedures in the art. Forexample, laccases may be recovered from a culture medium as described inU.S. Publication No. 2008/0196173. In some embodiments, the enzyme isexpressed intracellularly and is recovered following disruption of thecell membrane.

In an embodiment, the expression host may be Trichoderma reesei with thelaccase coding region under the control of a CBH1 promoter andterminator. (See, e.g., U.S. Pat. No. 5,861,271). The expression vectormay be pTrex3g, as disclosed in U.S. Pat. No. 7,413,887.

In some embodiments, laccases are expressed as described in U.S.Publication No. 2008/0196173 or U.S. Ser. No. 12/261,306.

In some embodiments, the laccases enzyme is laccase D from Cerrenaunicolor, e.g., as described in International Patent Publication No.WO08/076,322. In particular embodiments, the laccase has the amino acidsequence shown, below (SEQ ID NO:4):

AIGPVADLHIVNKDLAPDGVQRPTVLAGGTFPGTLITGQKGDNFQLNVIDDLTDDRMLTPTSIHWHGFFQKGTAWADGPAFVTQCPIIADNSFLYDFDVPDQAGTFWYHSHLSTQYCDGLRGAFVVYDPNDPHKDLYDVDDGGTVITLADWYHVLAQTVVGAATPDSTLINGLGRSQTGPADAELAVISVEHNKRYRFRLVSISCDPNFTFSVDGHNMTVIEVDGVNTRPLTVDSIQIFAGQRYSFVLNANQPEDNYWIRAMPNIGRNTTTLDGKNAAILRYKNASVEEPKTVGGPAQSPLNEADLRPLVPAPVPGNAVPGGADINHRLNLTFSNGLFSINNASFTNPSVPALLQILSGAQNAQDLLPTGSYIGLELGKVVELVIPPLAVGGPHPFHLHGHNFWVVRSAGSDEYNFDDAILRDVVSIGAGTDEVTIRFVTDNPGPWFLHCHIDWHLEAGLAIVFAEGINQTAAANPTPQAWDELCPKYNGLSASQKVKPKKGTAI

In some embodiments, the laccase enzyme includes is at least about 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even99.5% identical to the amino acid sequence set forth in SEQ ID NO: 4.

Suitable laccase enzyme systems may include chemical mediator agentswhich enhance the activity of the laccase enzyme. Such mediators act asa redox mediators to effectively shuttle electrons between the enzymeexhibiting oxidase activity and a dye, pigment (e.g., indigo),chromophore (e.g., polyphenolic, anthocyanin, or carotenoid, forexample, in a colored stain), or other secondary substrate or electrondonor. Chemical mediators are elsewhere referred to as enhancers andaccelerators.

The mediator may be a phenolic compound, for example, methyl syringate,and related compounds, as described in PCT Application Nos. WO95/01426and WO96/12845. The chemical mediator may also be an N-hydroxy compound,an N-oxime compound, or an N-oxide compound, for example,N-hydroxybenzotriazole, violuric acid, or N-hydroxyacetanilide. Thechemical mediator may also be a phenoxazine/phenothiazine compound, forexample, phenothiazine-10-propionate. The chemical mediator may furtherbe 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Otherchemical mediators are well known in the art. For example, the compoundsdisclosed in PCT Application No. WO95/01426 are known to enhance theactivity of a laccase. In some embodiments, the mediator may beacetosyringone, methyl syringate, ethyl syringate, propyl syringate,butyl syringate, hexyl syringate, or octyl syringate.

In some embodiments, the mediator is 4-cyano-2,6-dimethoxyphenol,4-carboxamido-2,6-dimethoxyphenol or an N-substituted derivative thereofsuch as, for example, 4-(N-methyl carboxamido)-2,6-dimethoxyphenol,4-[N-(2-hydroxyethyl)carboxamido]-2,6-dimethoxyphenol, or4-(N,N-dimethyl carboxamido)-2,6-dimethoxyphenol.

In some embodiments, the mediator is described by the following formula:

in which formula A is a group such as —R, -D, —CH═CH-D, —CH═CH—CH═CH-D,—CH═N-D, —N═N-D, or —N═CH-D, in which D is selected from the groupconsisting of —CO-E, —SO₂-E, —CN, —NXY, and —N⁺XYZ, in which E may be—H, —OH, —R, —OR, or —NXY, and X and Y and Z may be identical ordifferent and selected from —H, —OH, —OR and —R; R being a C₁-C₁₆ alkyl,preferably a C₁-C₈ alkyl, which alkyl may be saturated or unsaturated,branched or unbranched and optionally substituted with a carboxy, sulfoor amino group; and B and C may be the same Or different and selectedfrom C_(m)H_(2m)+₁; 1≦m≦5.

In some embodiments, A in the above mentioned formula is —CN or —CO-E,in which E may be —H, —OH, —R, —OR, or —NXY, where X and Y may beidentical or different and selected from —H, —OH, —OR and —R, R being aC₁-C₁₆ alkyl, preferably a C₁-C₈ alkyl, which alkyl may be saturated orunsaturated, branched or unbranched and optionally substituted with acarboxy, sulfo or amino group; and B and C may be the same or differentand selected from C_(m)H_(2m+1); 1≦m≦5. In one embodiment, the mediatoris 4-hydroxy-3,5-dimethoxybenzonitrile (also termed “syringonitrile” or“SN” interchangeably herein). A may be placed meta to the hydroxy groupinstead of being placed in the para-position, as shown.

For textile processing applications, the mediator may be present in aconcentration of about 0.005 to about 1000 mmole per g textile, e.g.,denim, about 0.05 to about 500 mmole per g textile, about 0.1 to about100 mmole per g textile, about 1 to about 50 μmole per g textile, orabout 2 to about 20 μmole per g textile.

The mediators may be prepared by methods known to the skilled artisan,such as those disclosed in PCT Application Nos. WO97/11217 and WO96/12845 and U.S. Pat. No. 5,752,980. Suitable mediators for use hereinare described, for example, in U.S. Publication No. 2008/0189871.

Desizing Enzymes

The present compositions and methods for abrading and color modificationmay be used in combination with enzymatic desizing. Desizing istypically performed prior to abrading and to color modification. One ormore desizing enzymes may be used.

In some embodiments, the desizing enzyme is an amylolytic enzyme, suchas an α-amylase, a β-amylase, a mannanases, a glucoamylases, or acombination thereof.

Suitable α and β-amylases include those of bacterial or fungal origin,as well as chemically or genetically modified mutants and variants ofsuch amylases. Suitable α-amylases include α-amylases obtainable fromBacillus species. Suitable commercial amylases include but are notlimited to OPTISIZE® 40, OPTISIZE® 160, OPTISIZE® HT 260, OPTISIZE® HT520, OPTISIZE® HT Plus, OPTISIZE® FLEX (all from Genencor), andDURAMYL™, TERMAMYL™, FUNGAMYL™ and BAN™ (all available from NovozymesA/S, Bagsvaerd, Denmark). Other suitable amylolytic enzymes include theCGTases (cyclodextrin glucanotransferases, EC 2.4.1.19), e.g., thoseobtained from species of Bacillus, Thermoanaerobactor orThermoanaero-bacterium.

The activity of OPTISIZE® 40 and OPTISIZE® 160 is expressed in RAU/g ofproduct. One RAU is the amount of enzyme which will convert 1 gram ofstarch into soluble sugars in one hour under standard conditions. Theactivity of OPTISIZE® HT 260, OPTISIZE® HT 520 and OPTISIZE® HT Plus isexpressed in TTAU/g. One TTAU is the amount of enzyme that is needed tohydrolyze 100 mg of starch into soluble sugars per hour under standardconditions. The activity of OPTISIZE® FLEX is determined in TSAU/g. OneTSAU is the amount of enzyme needed to convert 1 mg of starch intosoluble sugars in one minute under standard conditions.

The precise dosage of the amylase varies depending on the process type.Smaller dosages would require more time than larger dosages of the sameenzyme. However, there is no upper limit on the amount of desizingamylase other than what may be dictated by the physical characteristicsof the solution. Excess enzyme does not hurt the fabric; it allows for ashorter processing time. Based on the foregoing and the enzyme utilizedthe following minimum dosages for desizing are suggested:

Minimum dosage Typical Range (per liter of (per liter of Amylase Productdesizing liquor) desizing liquor) OPTISIZE ® 40 1,000 RAU 2,000-70,000RAU OPTISIZE ® 160 1,000 RAU 2,000-70,000 RAU OPTISIZE ® HT 260 1,000TTAU 3,000-100,000 TTAU OPTISIZE ® HT 520 1,000 TTAU 3,000-100,000 TTAUOPTISIZE ® HT Plus 1,000 TTAU 3,000-100,000 TTAU OPTISIZE ® FLEX 5,000TSAU 13,000-65,000 TSAU

The desizing enzymes may be derived from the enzymes listed above inwhich one or more amino acids have been added, deleted, or substituted,including hybrid polypeptides, so long as the resulting polypeptidesexhibit desizing activity. Such variants useful in practicing thepresent invention can be created using conventional mutagenesisprocedures and identified using, e.g., high-throughput screeningtechniques such as the agar plate screening procedure.

The desizing enzyme is added to the aqueous solution (i.e., the treatingcomposition) in an amount effective to desize the textile materials.Typically, desizing enzymes, such as α-amylases, are incorporated intothe treating composition in amount from about 0.00001% to about 2% ofenzyme protein by weight of the fabric, preferably in an amount fromabout 0.0001% to about 1% of enzyme protein by weight of the fabric,more preferably in an amount from about 0.001% to about 0.5% of enzymeprotein by weight of the fabric, and even more preferably in an amountfrom about 0.01% to about 0.2% of enzyme protein by weight of thefabric.

Catalase

In some embodiments, a catalase enzyme may be used to catalyze thedecomposition of residual hydrogen peroxide as any stage of textileprocessing. Catalase is routinely used for “bleach clean-up,” whichbroadly refers to the destruction of residual hydrogen peroxide used tobleach (i.e., whiten and brighten) textiles prior to dying. Catalase isalso routinely used for the destruction of hydrogen peroxide used todecolorize residual dyes present in aqueous dying solutions. Catalasemay also be used to destroy residual hydrogen peroxide from theperhydrolase system. Catalase for bleach clean-up and to for destroyresidual hydrogen peroxide from the perhydrolase system may be addeddirectly to the bath without rinsing.

Exemplary catalase enzymes are Catalase T100 and OXY-GONE® T400,available from Genencor, and CATAZYME® or TERMINOX® Ultra, availablefrom Novozymes. An exemplary catalase is described in European PatentNo. EP 0 629 134.

Additional Enzymes

It will be appreciated that one or more cellulase, perhydrolase,laccase, amylase, mannanase, catalase, or other enzyme mentioned,herein, may be used in the present compositions and methods. Moreover,any number of additional enzymes (or enzyme systems) can be combinedwith the present compositions and methods without defeating the spiritof the disclosure. Exemplary additional enzymes include but are notlimited to pectate lyases, pectinases, xylanases, polyesterases, andother enzymes that have been described and/or used for textileprocessing.

Methods

In some aspects, the present compositions and methods relate toenzymatic textile abrading and color modification using cellulase incombination with a perhydrolase system, in the same bath, without theneed to wash or rinse the textiles between enzymatic treatments.Abrading and color modification can be performed sequentially orsimultaneously. Abrading may be performed before or after colormodification. For the purpose of manufacturing indigo or sulfur-dyeddenim products, abrading (e.g., enzymatic “stonewashing”) usingcellulase is typically performed prior to color modification using aperhydrolase system.

In other embodiments, the present compositions and methods relate toenzymatic textile abrading and color modification using cellulase incombination with a perhydrolase system and a laccase system. Asdescribed herein, abrading using cellulase and color modification usinga perhydrolase system can be performed sequentially or simultaneously,in the same bath. As described in WO2010075402, abrading using cellulaseand color modification using a laccase system can also be performedsequentially or simultaneously, in the same bath. However, it has alsobeen discovered that the sequential use of a perhydrolase system and alaccase system, in either order, allows a textile manufacturer toproduce a vast array of different textile finishes and colors using onlya limited suite of enzyme systems.

Exemplary finishes and colors for indigo-dyed denim that can be obtainedusing various embodiments of the present compositions and methods arelisted in the Tables shown in FIGS. 1-5. The exemplary cellulase used toobtain the indicated effects was MEX-500; however, as described in theappended Examples, other acid and neutral cellulases can be used withsimilar results. In particular embodiments, sulfur-dyed textiles can beprocessed to impart a grey cast without producing a brown tint. Theexemplary perhydrolase and laccase enzyme systems were PRIMAGREEN®EcoWhite 1 and PRIMAGREEN® EcoFade LT, respectively, although theseexemplary systems are also non-limiting. The particular finishes andcolors obtained with each exemplary process are less important than thefact that a wide array of different effects can be obtained using alimited number of enzymatic processes that are suitable for use insingle-bath combinations.

Although mainly exemplified using indigo and sulfur-dyed textiles, thepresent methods can be used color-modify textiles dyed with a largenumber of dyes. Examples of dyes include, but are not limited to, azo,monoazo, disazo, nitro, xanthene, quinoline, anthroquinone,triarylmethane, paraazoanyline, azineoxazine, stilbene, aniline, andphthalocyanine dyes, or mixtures thereof. In one embodiment, the dye isan azo dye (e.g., Reactive Black 5 (2,7-naphthalenedisulfonic acid,4-amino-5-hydroxy-3,6-bis((4-((2-(sulfooxy)ethyl)sulfonyl)phenyl)azo)-tetrasodiumsalt), Reactive Violet 5, methyl yellow, Congo red). In someembodiments, the dye is an anthraquinone dye (e.g., remazol blue),indigo (indigo carmine), a triarylmethane/paraazoanyline dye (e.g.,crystal violet, malachite green), or a sulfur-based dye. In variousembodiments, the dye is a reactive, direct, disperse, or pigment dye. Insome embodiments, the dye is a component of an ink.

One class of dyes that may be oxidatively color-modified usingenzymatically is the reactive dyes. Reactive dyes are chromophores thatinclude an activated or activatable functional group capable ofchemically interacting with the surface of an object to be dyed, such asa textile surface. Such interaction may take the form of a covalentbond. Exemplary functional groups include monochlorotriazine,monofluorochlorotriazine, dichlorotriazine, difluorochloropyrimidine,dichloroquinoxaline, trichloropyrimidine, vinyl amide, vinyl sulfone,and the like. Reactive dyes may have more than one functional group(e.g., bifunctional reactive dyes), thereby enabling a higher degree offixation to a fabric.

Combined with enzymatic desizing and enzymatic bleach clean-up using anenzyme such as catalase, the present compositions and methods representa complete enzymatic textile processing solution that allows a textilemanufacturer to produce textile products with an array of differentfinishes and colors, using only a limited number of enzyme systems.

Compositions and Kits of Parts

In another aspect, kits of parts are provided for performing thedescribed methods. Such kits include, for example, (i) a single-bathabrading and color modification kit, comprising a cellulase and aperhydrolase system, (ii) a color modification kit, comprising aperhydrolase system and a laccase system, (iii) an abrading and colormodification kit, comprising a cellulase, a perhydrolase system, and alaccase system, or (iv) complete enzymatic textile processing systems,which may further comprise a desizing enzyme, a catalase, a pectatelyase, or other enzymes listed herein or known in the art for use intextile processing. It will be appreciated that one or more enzymes ofeach type may be included in the kit.

The perhydrolase system may include a perhydrolase enzyme, a substratefor the perhydrolase enzyme, and a hydrogen peroxide source, in amountsand in ratios suitable for textile color modification. The laccaseenzyme system may include a laccase enzyme and a mediator in amounts andin ratios suitable for textile color modification.

Instructions for use may be provided in printed form or in the form ofan electronic medium such as a floppy disc, CD, or DVD, or in the formof a website address where such instructions may be obtained.

These and other aspects and embodiments of the present compositions andmethod will be apparent to the skilled person in view of the presentdescription. The following examples are intended to further illustrate,but not limit, the compositions and methods.

EXAMPLES

The following enzyme nomenclature is used in the Examples:

Trade name Description PRIMAGREEN ® Commercially available compositioncomprising the EcoWhite 1 S54V variant of Mycobacterium smegmatisperhydrolase PRIMAGREEN ® Commercially available composition comprisingEcoFade LT Cerrena unicolor laccase OPTISIZE ® Commercially availablecomposition comprising 160 amylase Bacillus amyloliquefaciens amylaseINDIAGE ® Commercially available composition comprising Neutra LStreptomyces sp. 11AG8 endoglucanase INDIAGE ® Commercially availablecomposition comprising a 2XL blend of Trichoderma reesei cellulasesPRIMAFAST ® Commercially available composition comprising a 200cellulase from Trichoderma reesei STCE Cellulase from Staphylotrichumcoccosporum MEX-500 Cellulase from Humicola insolens

Example 1 Effect of Perhydrolase Concentration on Color Modification ofIndigo-dyed Denim Materials

Perhydrolase (PRIMAGREEN® EcoWhite 1 (321 U/g), available from GenencorDivision, Danisco US, Inc.), was used in this experiment. H₂O₂ (30 wt %,analysis grade) and propylene glycol diacetate (PGDA, >99.7%) werepurchased from Sigma Aldrich.

Procedure

12 denim legs (ACG denim style 80270), weighing approximately 3 kg, weredesized in a Unimac UF 50 front loader rotary washing machine under thefollowing conditions:

-   -   Desizing for 15 minutes at 10:1 liquor ratio 50° C. with 0.5 g/l        (15 g) of OPTISIZE® 160 amylase (Genencor) and 0.5 g/l (15 g) of        a non-ionic surfactant (ULTRAVON® RW (Huntsman)).    -   2 cold rinse steps for 5 minutes at 30:1 liquor ratio.

Following desizing, the denim legs were stonewashed in a Unimac UF 50washing machine according to the following program:

-   -   Cold rinsing for 5 minutes at 10:1 liquor ratio    -   Stonewashing for 60 minutes at 10:1 liquor ratio at 55° C. with        1 kg of pumice stone, pH 6.5-7 (1 g/l of disodium        phosphate.2H₂O+0.53 g/l of citric acid H₂O) and 0.025 g/l of        MEX-500 neutral cellulase (Meiji Corp., Nagoya, Japan).    -   2 cold rinse steps of 5 min each.

The denim was dried in a household dryer and then used to make swatches(7×7 cm).

After stonewashing, the experiments were performed in a Launder-O-meter(Rapid Laboratory Dyeing Machine type H12) according to the followingprocess:

-   -   450 ml stainless steel reaction vessels were filled with 100 ml        of pH 8 phosphate buffer (8.9 g/l of disodium phosphate.2H₂O+0.4        g/l of monosodium phosphate anhydrous).    -   To each vessel five (7×7 cm) stonewashed denim swatches of 10 g        weight were added.    -   6 ml/l of H₂O₂ solution (30% wt) and 2 ml/l of PGDA (>99.7%) was        added.    -   Perhydrolase was added at concentrations of 0.01, 0.05, 0.3,        1.0, 3.0, or 10 ml/l.    -   The reaction vessels were closed and loaded into the        launder-O-meter, which was pre-heated to 60° C.    -   Incubation was performed for 60 minutes, after which the        swatches were rinsed by overflow, spun dry in an AEG IPX4        centrifuge, and dried with an Elna Press Electronic iron at        program cotton and evaluated.

Evaluation of Denim Swatches

The denim swatches were evaluated after perhydrolase treatment with aMinolta Chromameter CR 310 in the CIE Lab color space with a D 65 lightsource. Measurements were performed before and after perhydrolasetreatment and the results from five swatches were averaged. The totalcolor difference (TCD) was calculated using the formula:TCD=√(ΔL)²+(Δa)²+(Δb)². The results are shown in Table 1.

TABLE 1 Perhydrolase concentration (ml/l) TCD ΔL/Δa/Δb Buffer 0.440.41/0.13/0.10 0.01 0.56 0.40/0.32/−0.23 0.05 1.46 1.10/0.31/−0.90 0.31.97 1.50/0.34/−1.23 1 2.11 1.37/0.51/−1.52 3 2.05 1.41/0.41/−1.43 101.49 1.19/0.42/−0.80

These results demonstrate that the perhydrolase enzyme system canproduce a cast modification on dyed-textiles over a range of enzymeconcentrations.

Example 2 Effect of H₂O₂ and PGDA Concentrations on Color Modificationof Indigo-Dyed Denim Procedure

12 denim legs (ACG denim style 80270), weighing approximately 3 kg, weredesized and stonewashed as described in Example 1. After stonewashing,the experiments were performed in a Launder-O-meter (Rapid LaboratoryDyeing Machine type H12) according to the following process:

-   -   450 ml stainless steel reaction vessels were filled with 100 ml        of pH 8 phosphate buffer (8.9 g/l of disodium phosphate.2H₂O+0.4        g/l of monosodium phosphate anhydrous).    -   To each vessel five (7×7 cm) stonewashed denim swatches of 10 g        weight were added.    -   H₂O₂ solution (30% wt) and PGDA (>99.7%) were added according to        the experimental design as shown in Table 2.1.

TABLE 2.1 [H₂O₂] (ml/l) [PGDA] (ml/l) 7.55 3.8 15 7.5 0.1 7.5 7.55 3.80.1 0.1 15 0.1 7.55 3.8 6.0 3.0 0 3.0 6.0 0 15 3.8 7.55 7.5

-   -   1.0 ml/l of perhydrolase was added (PRIMAGREEN® EcoWhite 1 (321        U/g)).    -   The reaction vessels were closed and loaded into the        Launder-O-Meter which was pre-heated to 60° C.    -   Incubation was performed for 60 minutes, after which the        swatches were rinsed by overflow, spun dry in an AEG IPX4        centrifuge, and dried with an Elna Press Electronic iron at        program cotton, and evaluated.

Evaluation of Denim Swatches

The denim swatches were evaluated after perhydrolase treatment with aMinolta Chromameter CR 310 in the CIE Lab color space with a D 65 lightsource. Measurements were performed before and after perhydrolasetreatment and the results from five swatches were averaged. The totalcolor difference (TCD) was calculated using the formula:TCD=√(ΔL)²+(Δa)²+(Δb)². The results are shown in Table 2.2.

TABLE 2.2 [H₂O₂] (mm) [PGDA] (ml/l) TCD ΔL/Δa/Δb 7.55 3.8 2.331.03/0.36/−1.24 15 7.5 2.48 1.11/0.40/−1.37 0.1 7.5 1.09 0.57/0.02/0.007.55 3.8 2.31 1.04/0.45/−1.17 0.1 0.1 0.76 0.07/−0.04/−0.06 15 0.1 1.480.66/0.12/−0.49 6.0 3.0 2.55 1.50/0.24/−1.17 0 3.0 0.62 0.15/−0.06/0.226.0 0 0.80 −0.22/0.10/−0.15 15 3.8 2.17 0.62/0.43/−1.28 7.55 7.5 2.371.17/0.35/−1.19

These results demonstrate that the perhydrolase enzyme system canproduce a cast modification on dyed-textiles over a range of hydrogenperoxide and PGDA concentrations.

Example 3 Effect of Time on Color Modification of Indigo-Dyed DenimProcedure

12 denim legs (ACG denim style 80270), weighing approximately 3 kg, weredesized and stonewashed as described in Example 1. After stonewashing,the experiments were performed in a Launder-O-meter (Rapid LaboratoryDyeing Machine type H12) according to the following process.

-   -   450 ml stainless steel reaction vessels were filled with 100 ml        of pH 8 phosphate buffer (8.9 g/l Disodium phosphate.2H₂O+0.4        g/l Monosodium phosphate anhydrous).    -   To each vessel five (7×7 cm) stonewashed denim swatches of 10 g        weight were added.    -   6 ml/l of H₂O₂ solution (30% wt) and 0.2 ml/l of PGDA (>99.7%)        were added. 11.0 g/l of perhydrolase was added (PRIMAGREEN®        EcoWhite 1 (321 U/g)).    -   The reaction vessels were closed and loaded into the        Launder-O-Meter, which was pre-heated to 60° C.    -   Incubation was performed for 10, 20, 30, 40, 50, or 60 minutes,        after which the swatches were rinsed by overflow, spun dry in an        AEG IPX4 centrifuge, dried with an Elna Press Electronic iron at        program cotton, evaluated.

Evaluation of Denim Swatches

The denim swatches were evaluated after perhydrolase treatment with aMinolta Chromameter CR 310 in the CIE Lab color space with a D 65 lightsource. Measurements were performed before and after perhydrolasetreatment and the results from five swatches were averaged. The totalcolor difference (TCD) was calculated using the formula:TCD=√(ΔL)²+(Δa)²+(Δb)². The results are shown in Table 3.

TABLE 3 Time TCD ΔL/Δa/Δb (buffer) 1.09 1.05/0.27/0.05 10 1.480.97/0.30/−1.08 20 2.17 1.51/0.45/−1.49 30 2.05 1.28/0.53/−1.51 40 2.241.57/0.44/−1.55 50 2.45 1.80/0.49/−1.59 60 2.62 1.99/0.46/−1.64

These results demonstrate that the perhydrolase enzyme system canproduce a cast modification on dyed-textiles in a time-dependent manner.

Example 4 Effect of Temperature on Color Modification of Indigo-DyedDenim Procedure

12 denim legs (ACG denim style 80270), weighing approximately 3 kg, wasdesized and stonewashed as described in Example 1. After stonewashing,the experiments were performed in a Launder-O-meter (Rapid LaboratoryDyeing Machine type H12) according to the following process.

-   -   450 ml stainless steel reaction vessels were filled with 100 ml        of pH 8 phosphate buffer (8.9 g/l of disodium phosphate.2H₂O+0.4        g/l of monosodium phosphate anhydrous).    -   To each vessel five (7×7 cm) stonewashed denim swatches of 10 g        weight were added.    -   6 ml/l of H₂O₂ solution (30% wt) and 2 ml/l of PGDA (>99.7%) was        added.    -   1.0 ml/l of perhydrolase was added (PRIMAGREEN® EcoWhite 1 (321        U/g)).    -   The reaction vessels were closed and loaded into the        Launder-O-Meter, which was pre-heated to 30, 40, 50 or 60° C.    -   Incubation was performed for 60 minutes, the swatches rinsed by        overflow, spun dry in an AEG IPX4 centrifuge, dried with an Elna        Press Electronic iron at program cotton, evaluated.

Evaluation of Denim Swatches

The denim swatches were evaluated after perhydrolase treatment with aMinolta Chromameter CR 310 in the CIE Lab color space with a D 65 lightsource. Measurements were performed before and after perhydrolasetreatment and the results from five swatches were averaged. The totalcolor difference (TCD) was calculated using the formula:TCD=√(ΔL)²+(Δa)²+(Δb)². The results are shown in Table 4.

TABLE 4 Temperature ° C. TCD ΔL/Δa/Δb 30 (buffer only) 0.930.91/0.07/0.16 30 1.36 1.20/0.28/−0.57 40 (buffer only r) 0.780.77/0.11/−0.02 40 1.55 1.26/0.28/−0.86 50 (buffer only) 1.071.06/0.11/−0.02 50 2.02 1.63/0.32/−1.14 60 (buffer only) 0.90.86/0.24/−0.15 60 2.21 1.67/0.44/−1.38

These results demonstrate that the perhydrolase enzyme system canproduce a cast modification on dyed-textiles under different temperatureconditions.

Example 5 Abrading and Color Modification of Indigo-Dyed Denim using aSequential Cellulase-Perhydrolase Process Procedure

12 denim legs (ACG denim style 80270), weighing approximately 3 kg, weredesized in a Unimac UF 50 washing machine under the followingconditions:

-   -   Desizing for 15 minutes at 10:1 liquor ratio 50° C. with 0.5 g/l        (15 g) of OPTISIZE® 160 amylase (Genencor) and 0.5 g/l (15 g) of        a non-ionic surfactant (ULTRAVON® RW; Huntsman).    -   2 cold rinses for 5 minutes at 30:1 liquor ratio.

Following desizing, the denim was stonewashed in a Unimac UF 50 rotarywashing machine according to the following procedure:

-   -   Cold rinse for 5 minutes at 10:1 liquor ratio    -   Stonewashing for 60 minutes at 10:1 liquor ratio 55° C. with 1        kg of pumice stone, pH 4.8 (1 g/l of trisodium citrate 2        H₂O+0.87 g/l of citric acid H2O) 1.17 g/l of INDIAGE® 2XL        cellulase (Genencor)    -   2 cold rinse steps of 5 min each.    -   4 legs taken out as a control.

After stonewashing, treatment with perhydrolase was performed in aUnimac UF 50 washing machine according to the following process:

-   -   60 minutes at 10:1 liquor ratio, with 1 g/l perhydrolase        (PRIMAGREEN® EcoWhite 1 (321 U/g)), 6 g/l of H₂O₂ solution (30%        wt) and 3 g/l of PGDA (>99.7%) at pH 7 (1 g/l of disodium        phosphate.2 H₂O and 0.17 g/l of citric acid) and temperature of        60° C. The pH was maintained at 7 by adding 4 M of sodium        hydroxide solution.    -   2 cold rinses for 5 minutes at 30:1 liquor ratio.    -   The denim was dried in a household dryer.

Evaluation of Denim Legs

Bleaching of denim legs was evaluated after treatment with a MinoltaChromameter CR 310 in the CIE Lab color space with a D 65 light source.For each denim leg, 8 measurements were taken and the results of the 12legs (96 measurements) were averaged. The results are shown in Table 5.

TABLE 5 Treatment L/a/b Perhydrolase treatment 36.3/−0.29/−15.17

These results demonstrate that the perhydrolase enzyme system canproduce a cast modification on dyed-textiles in a sequentialcellulase-perhydrolase process in a large-scale scale machine.

Example 6 Abrading and Color Modification of Indigo-dyed Denim using aSequential Cellulase-Laccase-Perhydrolase Process Procedure

Denim, 12 legs (ACG denim style 80270) weighing approximately 3 kg, wasdesized in a Unimac UF 50 washing machine under the followingconditions:

-   -   Desizing for 15 minutes at 10:1 liquor ratio 50° C. with 0.5 g/l        (15 g) of OPTISIZE® 160 amylase (Genencor) and 0.5 g/l (15 g) of        a non-ionic surfactant (ULTRAVON® RW (Huntsman).    -   2 cold rinses for 5 minutes at 30:1 liquor ratio.

Following desizing, the denim was stonewashed in a Unimac UF 50 rotarywashing machine according to the following procedure:

-   -   Cold rinse for 5 minutes at 10:1 liquor ratio    -   Stonewashing for 60 minutes at 10:1 liquor ratio 55° C. with 1        kg of pumice stone, pH 4.8 (1 g/l of trisodium citrate.2        H₂O+0.87 g/l of citric acid H₂O) and 1.17 g/l of INDIAGE® 2XL        (Genencor)    -   2 cold rinse steps of 5 min each.

After stonewashing, laccase treatment was performed in a Unimac UF 50washing machine according to the following process:

-   -   30 minutes at 10:1 liquor ratio, with 3 g/l of ready to use        PRIMAGREEN® EcoFade LT 100 (Genencor) laccase and laccase        mediator at pH 6 and temperature of 30° C.    -   2 cold rinses for 5 minutes at 30:1 liquor ratio.    -   The denim was dried in a household dryer.

After bleaching with laccase, treatment with perhydrolase was performedin a Unimac UF 50 washing machine according to the following process:

-   -   60 minutes at 10:1 liquor ratio, with 1 g/l of perhydrolase        (PRIMAGREEN® EcoWhite 1 (321 U/g)), 6 g/l of H₂O₂ solution (30%        wt) and 3 g/l of PGDA (>99.7%) at pH 8 (8.9 g/l disodium        phosphate.2H₂O+0.4 g/1 monosodium phosphate anhydrous) and        temperature of 60° C.    -   2 cold rinses for 5 minutes at 30:1 liquor ratio    -   The denim was dried in a household dryer

Evaluation of Denim Legs

Bleaching of denim legs was evaluated after laccase treatment and afterperhydrolase treatment with a Minolta Chromameter CR 310 in the CIE Labcolor space with a D 65 light source. For each denim leg, 8 measurementswere taken and the results of the 12 legs (96 measurements) wereaveraged. The results are shown in Table 6.

TABLE 6 Treatment L/a/b Laccase 40.5/−1.5/−12.1 Laccase + Perhydrolase44.4/−1.3/−15.2

These results demonstrate that the perhydrolase enzyme system can beused in combination with a laccase enzyme system to produce a differentcolor modification.

Example 7 Color Modification of Pure Indigo-dyed Denim usingPerhydrolase Materials

Perhydrolase (PRIMAGREEN® EcoWhite 1, 326 U/g, 1.5 mg enzyme protein/g)was used in this experiment. H₂O₂ (30 wt %, analysis grade) and PGDA(>99.7%) were purchased from Sigma Aldrich.

Procedure

12 denim legs weighing approximately 3 kg, was desized in a Unimac UF 50washing machine under the following conditions:

-   -   Desizing for 15 minutes at 10:1 liquor ratio 50° C. with 0.5 g/l        (15 g) of OPTISIZE® 160 amylase (Genencor) and 0.5 g/l (15 g) of        a non-ionic surfactant (ULTRAVON® RW) (Huntsman).    -   2 cold rinses for 5 minutes at 30:1 liquor ratio.

Following the desizing the denim was stonewashed in a Unimac UF 50rotary washing machine according to the following program:

-   -   Cold rinse for 5 minutes at 10:1 liquor ratio.    -   Stonewashing for 60 minutes at 10:1 liquor ratio 55° C. with 1        kg of pumice stone, pH 4.8 (1 g/l of trisodium citrate.2        H₂O+0.87 g/l of citric acid H₂O) 1.17 g/l of INDIAGE® 2XL        cellulase (Genencor).    -   2 cold rinse steps of 5 min each.    -   6 legs were taken out and dried for evaluation.

After stonewashing, treatment with perhydrolase was performed in aUnimac UF 50 washing machine according to the following process:

-   -   60 minutes at 10:1 liquor ratio, with 1 g/l of perhydrolase        (PRIMAGREEN® EcoWhite 1, 326 U/g, 1.5 mg enzyme protein/g), 6        g/l of H₂O₂ solution (30% wt) and 3 g/l of PGDA (>99.7%) at pH 8        (9.0 g/l disodium phosphate.2H₂O+0.3 g/1 monosodium phosphate        anhydrous) and temperature of 60° C.    -   2 cold rinses for 5 minutes at 30:1 liquor ratio.    -   The denim was dried in a household dryer

Evaluation of Denim Legs

Bleaching of denim legs was evaluated after laccase treatment and afterperhydrolase treatment with a Minolta Chromameter CR 310 in the CIE Labcolor space with a D 65 light source. For each denim leg, 8 measurementswere taken and the results of the 12 legs (96 measurements) wereaveraged. The results are shown in Table 7.

TABLE 7 Treatment L/a/b Stonewashing 23.52/1.45/−11.85 Stonewashing +perhydrolase 25.47/1.23/−13.49

These results demonstrate that the perhydrolase enzyme system canproduce a cast modification on pure indigo-dyed-textiles in a sequentialcellulase-perhydrolase process.

Example 8 Abrading and Color Modification of Denim using a Single-BathCellulase-Perhydrolase Process Procedure

Desized denim, (2 legs for evaluation+ballast), weighing approximately 3kg, was stonewashed in a Renzacci LX 22 rotary washing machine accordingto the following protocol:

-   40 minutes at 10:1 liquor ratio 50° C., pH 6.5 with 0.4% INDIAGE®    Neutra L cellulase (Batch No. 40105358001, 5,197 NPCNU/g    (Genencor)).-   After stonewashing 1 leg was taken out and dried for evaluation.-   Following stonewashing, without drain the bath, the denim was    treated with perhydrolase according to the following protocol:    -   40 minutes at 10:1 liquor ratio, with 1 g/l perhydrolase        (PRIMAGREEN® EcoWhite 1,326 U/g, 1.5 mg enzyme protein/g), 6 g/l        of H₂O₂ solution (30% wt) and 3 g/l of PGDA (>99.7%) at pH 11 (2        g/l of soda ash) and temperatures of 50° C.    -   2 cold rinses for 3 minutes    -   The denim was dried in an industrial dryer.

Evaluation of Denim Legs

Color adjustment of denim legs was evaluated after treatment withperhydrolase with a Minolta Chromameter CR 310 in the CIE Lab colorspace with a D 65 light source. For each denim leg, 6 measurements weretaken and the results were averaged. The results are shown in Table 8.

TABLE 8 Treatment L/a/b Stonewashing 28.45/0.97/−13.25 Stonewashing +perhydrolase 31.12/0.50/−14.14 in a single bath

These results demonstrate that the perhydrolase enzyme system can beused in a sequential, single-bath cellulase-perhydrolase process.

Example 9 Abrading and Color Modification of Denim Using a SequentialCellulase-Perhydrolase-Laccase Process Procedure

Desized denim, (2 legs for evaluation+ballast), weighing approximately 6kg, was mild stonewashed in a belly washer according to the followingprotocol:

-   -   Stonewashing for 40 minutes at 10:1 liquor ratio 50° C. pH 6.5        with 0.1% INDIAGE®Neutra L cellulase (Batch No. 40105358001,        5,197 NPCNU/g (Genencor)).    -   2 cold rinse steps of 3 min each

After stonewashing with cellulase, treatment with perhydrolase wasperformed in a belly washer according to the following process:

-   -   40 minutes at 15:1 liquor ratio, 1 g/l of perhydrolase, 6 g/l of        H₂O₂ solution (30% wt) and 3 g/l of PGDA (>99.7%) at pH 11 (2.0        g/l soda ash) and temperature of 50° C.    -   2 cold rinses for 3 minutes

After color adjustment, laccase treatment was performed in a bellywasher according to the following process:

-   -   40 minutes at 15:1 liquor ratio, with 1 g/l of the ‘ready to        use’ PRIMAGREEN® EcoFade LT 100 (Batch No. 780913616 6292        GLacU/g (Genencor)) laccase and laccase mediator at 40° C.    -   2 cold rinses for 3 minutes.    -   1 leg was take out for evaluation and dried in a industrial        dryer.

After bleaching with laccase, color adjustment treatment withperhydrolase was performed in a Belly washer according to the followingprocess:

-   -   40 minutes at 15:1 liquor ratio, 1 g/l of perhydrolase        (PRIMAGREEN® EcoWhite 1, 326 U/g, 1.5 mg enzyme protein/g), 6        g/l of H₂O₂ solution (30% wt) and 3 g/l of PGDA (>99.7%) at pH        11 (2.0 g/l soda ash) and temperature of 50° C.    -   2 cold rinses for 3 minutes    -   The denim was dried in a industrial dryer

Evaluation of Denim Legs

Bleaching and color adjustment of denim legs was evaluated after laccasetreatment and after perhydrolase treatment with a Minolta Chromameter CR310 in the CIE Lab color space with a D 65 light source. For each denimleg, 6 measurements were taken and the results were averaged. Theresults are shown in Table 9.

TABLE 9 Treatment L/a/b Cellulase + perhydrolase + 40.47/−1.28/−12.07laccase Cellulase + perhydrolase + 42.35/−1.02/−13.71 laccase +perhydrolase

These results demonstrate that the perhydrolase enzyme system can beused in different combinations with cellulase and a laccase enzymesystem, to produce unique finishing effects.

Example 10 Color Modification of Sulfur-Dyed Khaki Garments UsingPerhydrolase Materials

Perhydrolase (PRIMAGREEN® EcoWhite 1,326 U/g, 1.5 mg enzyme protein/g)was used in this experiment. H₂O₂ (30 wt %, analysis grade), PGDA(>99.7%) were purchased from Sigma Aldrich.

Procedure

Sulfur-dyed garments weighing approximately 2 kg, were stonewashed in aUnimac UF 50 rotary washing machine according to the following program:

-   -   Cold rinse for 5 minutes at 15:1 liquor ratio.    -   Stonewashing for 60 minutes at 15:1 liquor ratio 55° C., pH 4.8        (1 g/l of trisodium citrate.2 H₂O+0.87 g/l of citric acid H₂O)        1.0 g/l of INDIAGE® 2XL cellulase (Genencor).    -   2 cold rinse steps of 5 min each.    -   The garments were taken out and dried for evaluation.

After stonewashing, treatment with perhydrolase was performed in aUnimac UF 50 washing machine according to the following process:

-   -   60 minutes at 15:1 liquor ratio, with 1 g/l of perhydrolase        (PRIMAGREEN® EcoWhite 1,326 U/g, 1.5 mg enzyme protein/g), 6 g/l        of H₂O₂ solution (30% wt) and 3 g/l of PGDA (>99.7%) at 2 g/l of        sodium carbonate (pH 11) and temperature of 50° C.    -   2 cold rinses for 5 minutes at 30:1 liquor ratio.    -   The garments were dried in a household dryer

Evaluation of Denim Legs

Bleaching of denim legs was evaluated after perhydrolase treatment witha Minolta Chromameter CR 310 in the CIE Lab color space with a D 65light source. For each garment, 16 measurements were taken. The resultsare shown in Table 10.

TABLE 10 Treatment L/a/b Stonewashing 23.00/1.36/−1.18 Stonewashing +perhydrolase 26.72/1.44/−0.52

These results demonstrate that the perhydrolase enzyme system canproduce color modification on sulfur-dyed khaki garments.

Example 11 Abrading and Color Modification of Denim using a Single-BathAcid Cellulase-Perhydrolase Process Procedure

100% cotton, and 65% cotton/35% polyester sulfur-dyed legs weighingapproximately 5 kg, were desized in a twin belly washer YXG-80×2 underthe following conditions:

-   -   Desizing for 15 minutes at 15:1 liquor ratio 60° C. with 0.4 g/l        of OPTISIZE® 160 amylase (Genencor) and 0.5 g/l of a non-ionic        surfactant (ULTRAVON® RW; Huntsman).    -   2 cold rinse steps for 2 minutes at 30:1 liquor ratio.

Desized legs (4 legs 100% cotton sulfur-dyed and 4 legs 65% cotton/35%polyester sulfur-dyed+ballast), weighing approximately 5 kg, werestonewashed in twin belly washer YXG-80×2 according to the followingprotocol:

-   -   30 minutes at 15:1 liquor ratio 50° C., pH 4.7 (set with 20 ml        99.8% acetic acid) with 0.5 g/l PRIMAFAST® 200 cellulase from        Genencor.    -   After stonewashing 4 legs (2 legs 100% cotton sulfur-dyed and 2        legs 65% cotton/35% polyester sulfur-dyed) were taken out and        dried for evaluation

Following stonewashing, and without draining the bath, the legs weretreated with perhydrolase according to the following protocol:

-   -   60 minutes at 15:1 liquor ratio, with 1 g/l perhydrolase        (PRIMAGREEN® EcoWhite 1,326 U/g, 1.5 mg enzyme protein/g), 6 g/l        of H₂O₂ solution (30% wt) and 3 g/l of PGDA (>99.7%) at pH 7.6        (12 g/l of a blend 95% disodium phosphate dihydrate+5% of        monosodium phosphate anhydrous) and temperatures of 60° C.    -   2 cold rinses for 2 minutes.    -   The denim was dried in an industrial dryer.

Evaluation of Denim Legs

Color adjustment of the sulfur-dyed legs was evaluated after treatmentwith perhydrolase with a Minolta Chromameter CR 310 in the CIE Lab colorspace with a D 65 light source. For each denim leg, 4 measurements weretaken and the results were averaged. The results are shown in Table 11.

TABLE 11 L/a/b 100% cotton sulfur-dyed legs Stonewashing25.01/1.35/−1.86 Stonewashing + perhydrolase 35.25/1.49/−1.92 in asingle bath 65% cotton/35% polyester sulfur-dyed legs Stonewashing21.92/1.78/−2.28 Stonewashing + perhydrolase 33.65/1.76/−2.08 in asingle bath

These results demonstrate that the perhydrolase enzyme system canproduce color modification on sulfur-dyed 100% cotton and cotton blendmaterials, when used in combination with an acid cellulase in asingle-bath, cellulase-perhydrolase process.

Example 12 Abrading and Color Modification of Denim using a Single-BathNeutral Cellulase-Perhydrolase Process Procedure

100% cotton, and 65% cotton/35% polyester legs, weighing approximately 5kg, were desized in a twin belly washer YXG-80×2 under the followingconditions:

-   -   Desizing for 15 minutes at 15:1 liquor ratio 60° C. with 0.4 g/l        of OPTISIZE® 160 amylase (Genencor) and 0.5 g/l of a non-ionic        surfactant (ULTRAVON® RW; Huntsman).    -   2 cold rinse steps for 2 minutes at 30:1 liquor ratio.

Desized legs (4 legs 100% cotton sulfur-dyed and 4 legs 65% cotton/35%polyester sulfur-dyed+ballast), weighing approximately 5 kg, werestonewashed in twin belly washer YXG-80×2 according to the followingprotocol:

-   -   30 minutes at 15:1 liquor ratio 50° C., pH 7.3 (set with 65 ml        of 5% acetic acid solution) 0.1 g/l STCE cellulase (Meiji Corp.,        Nagoya, Japan).    -   After stonewashing 4 legs (2 legs 100% cotton sulfur-dyed and 2        legs 65% cotton/35% polyester sulfur-dyed) were taken out and        dried for evaluation

Following stonewashing, and without drain the bath, the legs weretreated with perhydrolase according to the following protocol:

-   -   60 minutes at 15:1 liquor ratio, with 1 g/l perhydrolase        (PRIMAGREEN® EcoWhite 1,326 U/g, 1.5 mg enzyme protein/g), 6 g/l        of H₂O₂ solution (30% wt) and 3 g/l of PGDA (>99.7%) at pH 7.6        (12 g/l of a blend 95% disodium phosphate dihydrate+5% of        monosodium phosphate anhydrous) and temperatures of 60° C.    -   2 cold rinses for 2 minutes.    -   The denim was dried in an industrial dryer.

Evaluation of Denim Legs

Color adjustment of the sulfur-dyed legs was evaluated after treatmentwith perhydrolase with a Minolta Chromameter CR 310 in the CIE Lab colorspace with a D 65 light source. For each denim leg, 4 measurements weretaken and the results were averaged. The results are shown in Table 12.

TABLE 12 L/a/b 100% cotton sulfur-dyed legs Stonewashing25.36/1.32/−1.92 Stonewashing + perhydrolase 36.75/1.49/−1.77 in asingle bath 65% cotton + 35% polyester sulfur-dyed legs Stonewashing21.83/1.79/−2.27 Stonewashing + perhydrolase 34.51/1.76/−1.99 in asingle bath

These results demonstrate that the perhydrolase enzyme system canproduce color modification on sulfur-dyed 100% cotton and cotton blendmaterials, when used in combination with a neutral cellulase in asingle-bath, cellulase-perhydrolase process.

Although the foregoing invention has been described in some detail byway of illustration and examples for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications may be practiced without departingfrom the spirit and scope of the invention. Therefore, the descriptionshould not be construed as limiting the scope of the invention.

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entireties for all purposesand to the same extent as if each individual publication, patent, orpatent application were specifically and individually indicated to be soincorporated by reference.

1. An enzymatic method for abrading and modifying the color of a dyedtextile, comprising: (a) contacting the textile with a cellulase toabrade the textile; and (b) contacting the textile with a perhydrolaseenzyme system to modify the color of the textile; wherein (a) and (b)are performed in a single bath.
 2. The method of claim 1, wherein (a)and (b) are performed sequentially or simultaneously.
 3. The method ofclaim 1, wherein (a) is preceded by an enzymatic desizing step.
 4. Themethod of claim 3, wherein the enzymatic desizing step is performed inthe same bath as (a) and (b).
 5. The method of claim 1, wherein (b) isfollowed by the addition of a catalase enzyme.
 6. The method of claim 5,wherein the catalase enzyme is added to the same bath in which (a) and(b) are performed.
 7. An enzymatic method for abrading and modifying thecolor of a dyed textile, comprising: (a) contacting the textile with acomposition comprising a cellulase to abrade the textile; (b) contactingthe textile with a laccase enzyme system to perform a first colormodification of the textile; and (c) contacting the textile with aperhydrolase enzyme system to perform a second color modification of thetextile; wherein the overall color modification produced by thecombination of (b) and (c) is different from the first colormodification in (b) and the second color modification in (c).
 8. Themethod of claim 7, wherein (b) is performed before (c).
 9. The method ofclaim 8, wherein (a) and (b) are performed sequentially orsimultaneously in a single bath.
 10. The method of claim 7, wherein (c)is performed before (b).
 11. The method of claim 10, wherein (a) and (c)are performed sequentially or simultaneously in a single bath.
 12. Themethod of claim 10 or 11, wherein (b) is followed by: (d) contacting thetextile with the perhydrolase enzyme system to perform a third colormodification of the dyed textile.
 13. The method of claim 7, wherein (a)is preceded by an enzymatic desizing step.
 14. The method of claim 13,wherein the enzymatic desizing step is performed in the same bath as(a).
 15. The method of claim 7, wherein (c) is followed by the additionof a catalase enzyme.
 16. The method of claim 7, wherein catalase enzymeis added to the same bath in which any of (a), (b), and/or (c) areperformed.
 17. The method of claim 1, wherein the cellulase is selectedfrom an acid cellulase, a neutral cellulase, and an alkaline cellulase.18. The method of claim 1, wherein the perhydrolase enzyme systemcomprises a perhydrolase enzyme and an ester substrate, wherein theperhydrolase enzyme catalyzes perhydrolysis of the ester substrate witha perhydrolysis:hydrolysis ratio equal to or greater than
 1. 19. Themethod of claim 1, wherein the perhydrolase enzyme system comprises aMycobacterium smegmatis perhydrolase or a variant, thereof.
 20. Themethod of claim 1, wherein the perhydrolase enzyme is a S54V variant ofMycobacterium smegmatis perhydrolase, or a variant, thereof.
 21. Themethod of claim 1, wherein the laccase enzyme is a Cerrena unicolorlaccase, or a variant, thereof.
 22. The method of claim 1, wherein thetextile is denim.
 23. The method of claim 1, wherein the dye is indigodye.
 24. The method of claim 1, wherein the dye is sulfur dye.
 25. Atextile produced by the method of claim
 1. 26. The textile of claim 25,wherein the textile is indigo-dyed denim.
 27. The textile of claim 25,wherein the textile is sulfur-dyed denim.